Evidence of two isomers of phosphatidylinositol in plant tissue

The structure of phosphatidylinositol in barley (Hordeum vulgare) aleurone layers was investigated by chemical degradation. In vivo myo-[2-³H]inositol-labeled phosphatidylinositol was first converted to glycerophosphoinositol and, subsequently, after removal of the glycerol moiety, to inositol monophosphate. Here, we present data that show that, in addition to the commonly occurring 1,2-diacylglycero-3-(d-myo-inositol-1-phosphate), barley aleurone cells contain a novel second isomer of phosphatidylinositol that differs in structure of the head group.     

The distribution of phosphatidylinositol in microsomal membranes from rat liver after biosynthesis de novo. Evidence for the existence of different pools of microsomal phosphatidylinositol by the use of phosphatidylinositol-exchange protein

1. The phosphatidylinositol-exchange protein from bovine brain was used to determine to what extent phosphatidylinositol in rat liver microsomal membranes is available for transfer. 2. The microsomal membranes used in the transfer reaction contained either phosphatidyl[2-³H]inositol or ³²P-labelled phospholipid. The ³²P-labelled microsomal membranes were isolated from rat liver after an intraperitoneal injection of [³²P]P_i. The ³H-labelled microsomal membranes and rough- and smooth-endoplasmic-reticulum membranes were prepared in vitro by the incorporation of myo-[2-³H]inositol into phosphatidylinositol by either exchange in the presence of Mn²⁺ or biosynthesis de novo in the presence of CTP and Mg²⁺. 3. Tryptic or chymotryptic treatment of the microsomes impaired the biosynthesis de novo of phosphatidylinositol. It was therefore concluded that the biosynthesis of phosphatidylinositol and/or its immediate precursor CDP-diacylglycerol takes place on the cytoplasmic surface of the microsomal membrane. 4. Under the conditions of incubation 42% of the microsomal phosphatidyl[2-³H]inositol was transferred with an estimated half-life of 5min; 38% was transferred with an estimated half-life of about 1h; the remaining 20% was not transferable. Identical results were obtained irrespective of the method of myo-[2-³H]inositol incorporation. 5. Both measurement of phosphatidylinositol phosphorus in the microsomes after transfer and the transfer of microsomal [³²P]phosphatidylinositol indicate that phosphatidyl[2-³H]-inositol formed by exchange or biosynthesis de novo was homogeneously distributed throughout the microsomal phosphatidylinositol. 6. We present evidence that the slowly transferable pool of phosphatidylinositol does not represent the luminal side of the microsomal membrane; hence we suggest that this phosphatidylinositol is bound to membrane proteins.     

Stimulation of the incorporation of 32Pi and myo-(2-3H)inositol into the phosphoinositides in polymorphonuclear leukocytes during phagocytosis

The effect of phagocytosis on the incorporation of ³²P_i and myo-[2-³H]inositol into the phosphoinositides (phosphatidylinositol, diphosphoinositide, and triphosphoinositide) by polymorphonuclear leukocytes from guinea pig peritoneal exudates has been studied. The results show that phagocytosis enhanced the incorporation of ³²P_i and myo-[2-³H]inositol into all three inositides in polymorphonuclear leukocytes. Pulse-chase experiments revealed that phagocytosis did not stimulate the loss of the label from the inositides. The findings indicate that the increased radioactivity of the phosphoinositides in polymorphonuclear leukocytes during phagocytosis is due to a greater rate of synthesis of these phospholipids at the time of labeling, rather than due to an increase in the rate of their turnover.     

Phosphatidylinositol(4,5)bisphosphate and Phosphatidylinositol(4)phosphate in Plant Tissues

Pea (Pisum sativum) leaf discs or swimming suspensions of Chlamydomonas eugametos were radiolabeled with [³H]myo-inositol or [³²P]Pi and the lipids were extracted, deacylated, and their glycerol moieties removed. The resulting inositol trisphosphate and bisphosphate fractions were examined by periodate degradation, reduction and dephosphorylation, or by incubation with human red cell membranes. Their likely structures were identified as d-myo-inositol(1,4,5)trisphosphate and d-myo-inositol(1,4,)-bisphosphate. It is concluded that plants contain phosphatidylinositol(4)phosphate and phosphatidylinositol(4,5)bisphosphate; no other polyphosphoinositides were detected.     

Metabolic Studies on Intermediates in the myo-Inositol Oxidation Pathway in Lilium longiflorum Pollen: III. Polysaccharidic Origin of Labeled Glucose

myo-Inositol-linked glucogenesis in germinated lily (Lilium longiflorum Thunb., cv. Ace) pollen was investigated by studying the effects of added l-arabinose or d-xylose on metabolism of myo-[2-³H]inositol and by determining the distribution of radioisotope in pentosyl and hexosyl residues of polysaccharides from pollen labeled with myo-[2-¹⁴C]inositol, myo-[2-³H]inositol, l-[5-¹⁴C]arabinose, and d-[5R,5S-³H]xylose.     

Increased labelling of polyphosphoinositide in chemically transformed cell line C3H10T1/2 CL8

The effect of malignant transformation of cells on phosphatidylinositol metabolism was investigated using C3H10T1/2 cells and its chemically transformed cell line, MCA CL-16 cells. We found that incorporation of [³²P]P_i into polyphosphoinositide was greatly increased in the transformed cells. A similar tendency was observed when myo-[2-³H]inositol was used as a labelling reagent. It is also observed that influx of labelled inorganic phosphate is enhanced 2-fold by the cell transformation. Therefore, promotion of polyphosphoinositide labelling in the transformed cell might be caused not only by the enhanced metabolism of phosphatidylinositol but also by the increased membrane permeability for radioactive labelling reagents.     

Polyphosphoinositides are present in plasma membranes isolated from fusogenic carrot cells

Fusogenic carrot cells grown in suspension culture were labeled 12 hours with myo-[2-³H]inositol. Plasma membranes were isolated from the prelabeled fusogenic carrot cells by both aqueous polymer two-phase partitioning and Renografin density gradients. With both methods, the plasma membrane-enriched fractions, as identified by marker enzymes, were enriched in [³H]inositol-labeled phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP₂). An additional [³H]inositol-labeled lipid, lysophosphatidylinositol monophosphate, which migrated between PIP and PIP₂ on thin layer plates, was found primarily in the plasma membrane-rich fraction of the fusogenic cells. This was in contrast to lysophosphatidylinositol which is found primarily in the lower phase, microsomal/mitochrondrial-rich fraction.     

Phosphatidylinositol 4,5-bisphosphate: Light-mediated breakdown in the vertebrate retina

Isolated frog ($\text{Rana}\text{Pipiens}$) retinas were labeled in the dark with either [³²P]PO₄-orthophosphate or $\text{myo}$-[2-³H]inositol for 2.5–4 hrs. After washing the retinas with cold buffer, they were exposed to brief flashes of light (5 secs or 15 secs) and their rod outer segments isolated. Upon separation of labeled phospholipids, a specific decrease in label in phosphatidylinositol 4,5-bisphosphate was observed, whereas there was no significant effect on the labeling of phosphatidylinositol 4-phosphate, phosphatidylinositol, or phosphatidic acid. These results are indicative of a light-activated phosphatidylinositol 4,5-bisphosphate-specific phospholipase C in frog rod outer segments.     

Metabolic Studies on Intermediates in the myo-Inositol Oxidation Pathway in Lilium longiflorum Pollen: I. Conversion to Hexoses

The myo-inositol oxidation pathway was investigated in regard to its role as a source of carbon for products of hexose monophosphate metabolism in germinated pollen of Lilium longiflorum Thunb., cv. Ace. myo-[2-¹⁴]Inositol and d-[1-¹⁴C]glucuronate had similar distributions of radioactivity, contributing about three times more label to polysaccharide-bound glucose than myo-[2-³H]inositol. In the course of glucogenesis label from the latter appeared as tritiated water in the medium. This exchange could be enhanced by supplying d-[5R,5S-³H]xylose instead of myo-[2-³H]inositol. When the former was administered, [³H]glucose was the only labeled sugar residue found in polysaccharide products. The soluble constituents of d-[5R,5S-³H]xylose-labeled pollen contained no traces of labeled xylose despite massive uptake and utilization.     

Kinetics of protein-mediated transfer of rat pancreatic microsomal phosphatidylinositol to liposomes

Pancreatic microsomes were isolated from fasted and pilocarpine-injected rats and the microsomal phosphatidylinositol radiolabelled with myo-[2-³H]inositol by isotopic exchange. A standard reaction mixture was established in which partially purified rat liver phosphatidylinositol exchange proteins sustain a maximal rate of phosphatidylinositol transfer from rat pancreatic microsomes to liposomes. Determination of the transfer kinetics shows (1) that pancreatic microsomal phosphatidylinositol is partitioned approximately equally between a non-exchangeable and a single exchangeable pool and (2) that cholinergic stimulation does not significantly change the relative sizes of the two pools nor the exchange half-life of the latter pool.     

The effect of phentolamine on synaptosomal phosphatidylinositol in experimental galactose toxicity

Abnormal myo-[2-³H]inositol incorporation into phosphatidylinositol has been found in phentolamine-treated synaptosomes that were isolated from the cerebral hemispheres of galactose toxic rats and incubated with [³³P]P_i and myo-[2-³H] inositol. In galactose toxic rats phentolamine-stimulated myo-[2-³H]inositol labeling of phosphatidylinositol was 70% greater than in normal animals. This enhanced labeling of synaptosomal phosphatidylinositol in galactose toxic rats during stimulation with phentolamine is in marked contrast to the depressed myo-inositol labeling of phosphatidylinositol reported with acetylcholine stimulation.     

Identification of 3-hydroxy-6-methyl-2H-pyran-2-one from pyrolysis of phosphoric acid-treated cellulosic materials

The hydrogen isotope-effect that occurs in vitro during myo-inositol 1-phosphate synthase-catalyzed conversion of d-[5-³H]glucose 6-phosphate into myo-[2-³H]inositol 1-phosphate has been used to compare the functional role of the nucleotide sugar oxidation-pathway with that of the myo-inositol oxidation-pathway in germinating lily pollen. Results reveal a significant difference between the ³H/¹⁴C ratios of glucosyl and galactosyluronic residues from pectinase-amyloglucosidase hydrolyzates of the 70 % ethanol-insoluble fraction of d-[5-³H, 1-¹⁴-C]glucose-labeled, germinating lily pollen. This isotope effect at C-5 of d-glucose that occurred during its conversion into d-galactosyluronic residues of pectic substance is not explained by loss of ³H when UDP-d-[5-³H, 1-¹⁴C]glucose is oxidized by UDP-d-glucose dehydrogenase from germinating lily pollen. The evidence obtained from this study favors a functional role for the myo-inositol oxidation pathway during in vivo conversion of glucose into galactosyluronic residues of pectin in germinating lily pollen.     

Phosphatidylinositol Cycle Metabolites in Samanea saman Pulvini

The major metabolites of the phosphatidylinositol cycle from extracts of [³²PO₄]- and [³H]-inositol-labeled Samanea saman pulvini were separated. The membrane localized phosphoinositides were separated by thin layer chromatography, identified by comparison with purified lipid standards, and quantitated based on incorporation of radiolabel. The ratio of radioactivity in phosphatidylinositol:phosphatidylinositol 4-phosphate:phosphatidylinositol 4,5-bisphosphate is about 32:8:1. The aqueous inositol phosphates were separated by anion exchange chromatography using conventional liquid chromatography and by high performance liquid chromatography (HPLC) and were identified by comparison with standards. Analysis by HPLC reveals that ³²P-labeled pulvini have inositol 1-phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate that co-migrate with red blood cell inositol phosphates, but ³H-inositol-labeled pulvini appear to have a variant profile.     

Substance P-Induced Reduction in the Initial Accumulation of Cytosolic myo-[3H]Inositol in Rat Parotid Acinar Cells Mediated by the NK1 Tachykinin Receptor

Abstract: Stimulation of rat parotid acinar cells by the tachykinin neurokinin (NK) 1 receptor agonist substance P (SP) resulted in a significant reduction in the initial accumulation of cytosolic myo-[³H]inositol. This effect was rapid, because a reduction of ～15% could be seen already at 30 s, with the maximal effect (～45%) being observed at 15 min. The response to SP stimulation Was temperature dependent, because at 4°C no reduction was found, jln addition, at 4°C, cytosolic myo-[³H]inositol represented only 10% of the labeled inositol accumulated at 37°C. The SP-induced reduct on in cytosolic ravo[³H]inositol accumulation was concentration dependent; the EC₅₀ obtained for SP was 5.8 ± 2.5 nM. Spantide [N Arg¹, D-Trp⁷⁹, Leu]SP), a SP antagonist, used at a concentration oif 10⁵ A/, gave a competitive shift of the dose-response curve to SP. Various tachykinins and their analogs were evaluated for their ability to reduce cytosolic mvo-[³H]inositol. [L-Pro⁹]SP and SP methyl ester, two highly selective agonists of NK1 receptors, reduced the initial accumulation of myo-H]inositol with EQo values of 2.3 and 67.0 nM, respectively. Long SP C-terminal fragments were more potent than shorter ones. SP N-terminal fragments and SP free acid were -without effect. [Pro⁷]NKB, a selective NKB analog, had no effect. The rank order of potency of mammalian tachykinins was SP > NKA > NKB. These findings and the close correlation between EC50 values and IC₅₀ values obtained in binding studies implicate the NK 1 receptor. In addition, stimulation of muscarinic receptors by carbachol alscp resulted in a reduction in level of cytosolic mjw-[³H]inositol, with this effect being reversed by atropine. Moreover, atropine was unable tjo alter the SP-induced reduction in cytosolic myo-[³H]inositol accumulation. Other neurotransmitters, such as glutamic acid, serotonin, chplecystokinin, neurotensin, bradykinin, and neuropeptide Y, were without effect on initial cytosolic myo-[³H]inositol accumulation. In conclusion, NK1 and muscarinic receptors seem to regulate the membrane transport of inositol in acinar cells of the rat parotid gland. Measurement of the initial accumulation of cytosolic myo-[³H]inositol in this tissue could profitably be adopted as a very simple, rapid, [sensitive, and specific biochemical procedure for screening the activity of potential agonists and antagonists at NK1 receptors.     

Phytate metabolism in Petunia pollen

Phytic acid has been detected in the anthers of young flower buds of Petunia hybrida, the amount increasing slowly as the flower develops until anther dehydration, when there was a more rapid increase in phytic acid content. In mature pollen, the phytic acid content was found to be 2.0 % by weight, of which 90 % was water soluble, while free myo-inositol was a relatively low 0.06 % by weight. Breakdown of phytic acid was initiated soon after pollen germination began, and its degradation products, myo-inositol and inorganic phosphate, were rapidly mobilized for phospholipid and pectin biosynthesis. Both are in high demand during pollen tube elongation. Utilization of myo-[2-³H]inositol for phospholipid biosynthesis was about five times that for pectin synthesis during the first few hours of pollen germination. The label in the phospholipid was identified as the myo-inositol moiety of phosphaltidylinositol, while the pectin material contained predominantly labelled arabinose, with smaller amounts of label in galacturonic acid, glucose and xylose. A chase experiment showed that the myo-inositol moiety of phosphatidylinositol was subject to a relatively rapid turnover, while the label in pectin was not. Labelling germinating pollen with [³²P]orthophosphate gave label in phosphatidic acid, phosphatidylinositol, phosphatidylethanolamine and phosphatidylcholine of the phospholipids. Phosphatidylinositol contained 30 % of this label initially, a proportion which declined to 10 % over longer periods of germination.     

Evidence for a Functional myo-Inositol Oxidation Pathway in Lilium longiflorum Pollen

Addition of myo-inositol to pentaerythritol-based germination media repressed the conversion of d-[1-¹⁴C]glucose to labeled uronosyl and pentosyl units of tube wall pectic substance in lily pollen (Lilium longiflorum Thunb.). Conversion of d-[1-¹⁴C]glucose to labeled glucosyl, galactosyl, and rhamnosyl units was unaffected. The reverse experiment, addition of d-glucose to pentaerythritol-based media, failed to affect the conversion of myo-[2-³H]inositol to uronosyl and pentosyl units although the flow of label into products of myo-inositol-linked glucogenesis was blocked. Results of these experiments are discussed in terms of a functional myo-inositol oxidation pathway.     

Phospholipid turnover in isolated rat pancreatic acini. Consideration of the relative roles of phospholipase A2 and phospholipase C

The purpose of the present study was to explore the interaction of phosphatidylinositol breakdown and the turnover of arachidonic acid in isolated rat pancreatic acini by using receptor agonists and the calcium ionophore ionomycin. Acini prelabelled with myo-[³H]inositol in vivo responded to carbachol with a rapid breakdown of phosphatidylinositol. In the presence of [³²P]P_i, carbachol increased labelling of phosphatidic acid and phosphatidylinositol within 1 and 5 min respectively. Carbachol also rapidly stimulated the incorporation of [¹⁴C]arachidonic acid into phosphatidylinositol within 2 min, and the peptidergic secretagogue caerulein caused the loss of radioactivity from phospholipids prelabelled with arachidonic acid. Ca²⁺ deprivation partially impaired the stimulatory action of carbachol on arachidonic acid turnover. In contrast with its stimulatory effects on [³²P]P_i and [¹⁴C]arachidonate incorporation, carbachol inhibited the incorporation of the saturated fatty acid stearic acid into phosphatidylinositol. Whereas ionomycin stimulation of phosphatidylinositol breakdown and [³²P]P_i labelling of phospholipids was slower in onset and less effective than carbachol stimulation, the ionophore effectively promoted (arachidonyl) phosphatidylinositol turnover within 2 min. These results implicate two separate pathways for stimulated phosphatidylinositol degradation in the exocrine pancreas, involving phospholipases A₂ and C. Whereas mobilization of cellular Ca²⁺ appears sufficient to cause activation of phospholipase A₂ and amylase secretion, additional events triggered by receptor activation may be required to act in concert with Ca²⁺ to optimally stimulate phospholipase C. The nature of the interaction between phospholipases A₂ and C and their specific physiological roles in pancreatic secretion remain to be elucidated.     

Human sodium/inositol cotransporter 2 (SMIT2) transports inositols but not glucose in L6 cells

To characterize the function of the sodium/inositol symporter SMIT2 in skeletal muscle, human SMIT2 cDNA was transfected into L6 myoblasts using pcDNA3.1 expression vector. Compared with the pcDNA3.1 vector only transfection, this overexpression increased the uptake of [³H]d-chiro-inositol (DCI) by 159-fold. [³H]myo-Inositol uptake increased by 37-fold. In contrast, [¹⁴C]d-glucose, [¹⁴C]2-deoxy-d-glucose, or [¹⁴C]3-O-methyl-d-glucose uptake remained unchanged in the presence of either 0, 5.5, or 25 mM unlabeled glucose. The K_m of DCI and myo-inositol for DCI uptake was 111.0 and 158.0 μM, respectively, whereas glucose competed for DCI uptake with a K_i of 6.1 mM. Insulin treatment of non-transfected L6 cells (2 μM for 24 h) increased [³H]DCI specific uptake 18-fold. DCI transport is up regulated by insulin and competitively inhibited by millimolar levels of glucose. Therefore, expression and/or function of SMIT2, a high affinity transporter specific for DCI and myo-inositol, may be reduced in diabetes mellitus, insulin resistance and polycystic ovary syndrome causing the abnormal DCI metabolism observed in these conditions.     

Effect of neomycin on phosphoinositide labelling and calcium binding in guinea-pig inner ear tissues in vivo and in vitro.

—Phospholipids of guinea-pig inner ear tissues were labelled in vivo by perilymphatic perfusion of the cochlea with [³²P]orthophosphate or myo-[³H]inositol. After 20-40 min the most highly labelled ³²P-lipids were phosphatidylinositol phosphate and diphosphate. Incorporation of [³H]inositol proceeded in the order phosphatidylinositol > phosphatidylinositol phosphate > phosphatidylinositol diphosphate. After treatment of animals with neomycin for 3 weeks, ³²P-incorporation into phosphatidylinositol diphosphate, but not into other lipids, was significantly decreased in the preparations of the organ of Corti and stria vascularis. In homogenates of inner ear tissues, the labelling of the polyphosphoinositides by [γ-³²P]ATP was increased and the hydrolysis of these lipids was blocked in the presence of 10^?4m -neomycin. Neomycin also competitively inhibited the binding of ⁴⁵Ca²⁺ to homogenates of these tissues.     

Epidermal growth factor stimulates the incorporation of phosphate into phosphatidic acid and phosphoinositides but does not affect phosphoinositide breakdown by phospholipase C in renal cortical slices

The effects of epidermal growth factor (EGF) on the metabolism of phosphatidic acid and phosphoinositides were examined using renal cortical slices labelled with either sodium [³²P]orthophosphate or myo-[³H]inositol. EGF was found to increase the incorporation of phosphate into phosphatidic acid and phosphoinositides. This effect is not dependent on external calcium and is inhibited by 12-O-tetradecanoylphorbol 13-acetate (TPA). When phospholipids were prelabelled, EGF did not decrease the level of ³²P in phosphatidic acid and phosphoinositides, and EGF did not affect the formation of inositol phosphates or the concentration of cAMP and cGMP in renal tissue. The results show that EGF stimulates the incorporation of phosphate into phosphatidic acid and phosphoinositides, but does not affect breakdown of phosphoinositides by phospholipase C in renal cortical slices.